Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Dyes that are able to preferentially bind to a specific biological ingredient in a sample enable the researcher to determine the presence or quantity of that specific ingredient. In addition, specific cellular structures can be monitored with respect to their spatial and temporal distribution in diverse environments. Furthermore, dyes can be used to determine ionic, electrical or metabolic properties of cellular organelles.
Mitochondria are the intracellular organelles responsible for aerobic metabolism in eukaryotic cells. Their abundance varies with cellular energy level and is a function of cell type, cell-cycle stage and proliferative state. There is a need in biology to detect and observe mitochondria particularly in cells, as a specific application or in conjunction with additional labeling of other components under study. Due to the strong proton gradient across the mitochondrial membrane (alternatively stated, the strong redox potential across the membrane) a variety of substances that possess a cationic charge have been found to selectively localize within functioning mitochondria. Under the proper conditions, this property has been used to localize a variety of fluorescent dyes within mitochondria for use in imaging (Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 1992, Set 30).
There are several "xanthylium" dyes that have proven useful for mitochondrial labeling. The fluorescent dyes tetramethylrosamine, rhodamine 123 and rhodamine 6G are readily sequestered in mitochondria, where localization of the dyes is a function of membrane potential. The reduced forms of these dyes are all colorless diaminodihydroxanthene derivatives, which become fluorescent only upon oxidation to the parent xanthylium dye within living cells. The fluorescent stain that results from intracellular oxidation can then be made to localize within the mitochondria. Furthermore, these mitochondrial stains have been used in conjunction with flow cytometry to sort and/or analyze cells. See, for example Rothe et al., JOURNAL OF IMMUNOLOGICAL METHODS, 138, 133 (1991), Martinez et al., EXP. CELL RES. 164, 551 (1986), Kliot-Fields et al. SOMATIC CELL GENETICS, 9, 375 (1983).
While currently available mitochondria stains, such as rhodamine 123 and tetramethylrosamine, are typically used to stain cells in concentrations of approximately 1-2 .mu.M, the dyes of the present invention provide bright mitochondrial staining at much lower concentrations. Typically concentrations between 20 nM and 500 nM are sufficient to give very good fluorescent staining of mitochondria.
All currently available xanthylium stains for mitochondria share a common drawback. The cationic dye is sequestered in the mitochondria in an equilibrium process, and mitochondrial staining can only be maintained by a functioning mitochondria. Attempts to fix stained cells generally result in cell death, the loss of mitochondrial potential, and therefore the loss of mitochondrial stain. Cells that are killed before staining do not stain well, again due to the lack of potential across the mitochondrial membrane. This is a drawback for researchers wishing to investigate mitochondrial function or viability in pathogenic species, who must choose between the very poor staining procedures available for fixed cells, or the additional hazards and costs associated with handling live pathogens. A method for assessing the viability of pathogenic species by staining with ethidium monoazide, then fixing, has been described (Riedy et al., CYTOMETRY, 12, 133 (1991). Staining with ethidium monoazide, however, only reveals the membrane permeability of the cell at the time of staining, and reveals no information regarding the metabolic state of the cell under investigation. The dyes of the present invention will indicate cells that possessed functioning mitochondria at the time of staining, even after fixation. The assessment of mitochondrial function is a much more meaningful indicator of metabolic activity than membrane permeability.
An additional drawback to the use of xanthylium mitochondrial stains is their incompatibility with the use of other labeling techniques, including immunocytochemical staining of intracellular antigens and in situ hybridization. The use of labeled antibodies, labeled oligonucleotides, or labeled probes for intracellular protein receptors require permeabilization of the cell membranes to allow the bulky labeling agents to enter the cellular space. During fixation and permeabilization, standard mitochondrial stains are washed away. Utilizing the dyes of the current invention, mitochondria can be stained, the cell can be fixed and permeabilized, and a variety of labeling agents can then be utilized for simultaneous visualization of mitochondria and other cellular components.
The dyes of the present invention are cationic fluorescent xanthylium dyes that localize within the mitochondria of living cells, and are retained there. The reduced diaminodihydroxanthene forms of the present dyes are oxidized intracellularly or within mitochondria to the fluorescent xanthylium form of the dyes, which then localize within the mitochondria. While previous examples of mitochondrial stains can localize within mitochondria, they are not retained as effectively as the dyes of the present invention. For example, cells that have been stained with rhodamine 123 can lose fluorescent staining in about 30 minutes when put into fresh medium that does not contain rhodamine 123. One of the most effective mitochondrial stains, tetramethylrosamine, maintains staining for only about 6-12 hours, depending on the concentration of the labeling solution used. In either case, essentially all mitochondrial staining is lost upon fixation of the sample cells. A major advantage of the present dyes lies in their long-term retention within the mitochondria, even through cell division, or after fixation and permeabilization of the sample cells. This retention is due to the presence of an alkylating group attached to the dye that reacts with intracellular nucleophiles, including thiols, to form a conjugate. This conjugate is retained within the mitochondria, and is not removed by washing or even after washing fixed and permeabilized cells.
The use of a haloalkyl functional group to retain a fluorescent reaction product of enzymatic activity within a cell has been described in the PCT Application USE OF HALOALKYL DERIVATIVES OF REPORTER MOLECULES TO ANALYZE METABOLIC ACTIVITY IN CELLS, by Haugland et al., Int. Publ. No. WO 93/04192. The reagents described in the reference contain a blocking group removed by the activity of a selected enzyme and a haloalkyl group that reacts with an intracellular thiol to retain the reagents in the cell. The reagents are substrates for selected enzymes and become fluorescent, or more fluorescent, after being acted upon by that enzyme. A haloalkyl functional group present on the substrate reacts with an intracellular thiol to give a fluorescent product that is retained within the cell even after washing or fixation. Exemplified and specifically described compounds of the above reference are retained within the cytoplasm of sample cells, but do not localize within the mitochondria of the cells. There is no indication that the compounds described in the reference localize in the mitochondria, or in any other organelle.
A specific haloalkyl derivative of a xanthylium reporter molecule, chloromethylbenzoyl-aminotetramethylrhodamine, is currently available from Molecular Probes Inc. (Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 1992, Set 24, under the trade name CellTracker.TM. Orange CMTMR). Unlike the mitochondria stains of the present invention, the overall charge of chloromethylbenzoyl-aminotetramethylrhodamine is neutral, and it does not localize in mitochondria (See FIG. 3).
It is an object of this invention to provide new fluorescent stains that are well-retained in mitochondria, particularly in the mitochondria of eukaryotic cells. It is further an object of this invention to use these materials to assess the mitochondrial function in eukaryotic cells, in particular, the assessment of mitochondrial function as it relates to the viability of eukaryotic cells. The assessment of mitochondrial function at the time of staining can then be carried out even after fixation of the cell. In conjunction with the mitochondrial stains of the invention, the mitochondria or cells can be stained with an additional detection reagent, such as a labeled antibody, labeled oligonucleotide, or other indicator for a specific cellular component or substructure.